Anticancer therapy

ABSTRACT

A subject afflicted with a cancer or precancerous condition is treated by administering an agent that increases expression of somatostatin receptors, and a cytotoxic recognition ligand. In an alternative embodiment, somatostatin analogs, which are radiolabeled are used to treat cancer or precancerous conditions.

CROSS-REFERENCES TO RELATED APPLICATIONS

This patent application is a division of U.S. patent application Ser.No. 15/427,355, filed Feb. 8, 2017, which is a continuation of U.S.patent application Ser. No. 14/669313, filed Mar. 26, 2015, which issuedas U.S. Pat. No. 9,610,371 on Apr. 4, 2017, which is a division of U.S.patent application Ser. No. 11/493,063, filed Jul. 26, 2006, nowabandoned, which claims priority to U.S. Provisional Patent ApplicationU.S. Ser. No. 60/703,810, filed Jul. 29, 2005, entitled “RadioisotopicTherapeutic Agent and Method” and U.S. Provisional Patent ApplicationU.S. Ser. No. 60/764,043, filed Jan. 31, 2006, entitled “CombinationAnticancer Therapy,” each of which applications is hereby incorporatedby reference in its entirety for all purposes.

GOVERNMENT FUNDING

The invention was made with government support under Grant No.DE-FG01-001NE23554. awarded by the United States Department of Energy,and Grant No. DHHS/PHS/NIH/NCRR/GCRC, MOI RR00997, awarded by theNational Institutes of Health National Center for Research Resources.The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Somatostatin is a 14-amino acid peptide hormone (somatostatin-14; SS-14)found in many cells, particularly those of neuroendocrine origin, thatacts as a neurotransmitter in the central nervous system. Reubi et al.,Cancer Res. 47, 5758-64 (1987). There is also a somatostatin variantreleased by β cells in the pancreatic islets that is a 28 amino acidpeptide (somatostatin-28; SS-28). Somatostatin has an inhibitory effecton growth hormone, and a generally antiproliferative effect.Somatostatin receptors (SSTR or SSR) are found on the surface of humantumor cells, including cells with amine precursor uptake anddecarboxylation properties, such as pituitary tumors, endocrinepancreatic tumors, carcinoids, paragangliomas, small cell lung cancers,medullary thyroid carcinomas and pheochromocytomas. Reubi et al.,Metabolism, 41, 104-10 (1992); Patel, Front. Neuroendocrinol. 20, 157-98(1999). Somatostatin receptors belong to the guanine nucleotide-bindingregulatory protein (G-protein)-linked receptor family.

Synthetic somatostatin analogs such as octreotide and lanreotide havebeen used for antitumor treatment and cancer detection. Jensen et at, J.Clin, Endocrinol. Metab., 85(10), 3507-8 (2000). Analogs of somatostatinwere developed because human somatostatin has a very short half-life incirculation (2-3 minutes) and is easily broken down by endogenouspeptidases. Rens-Domiano et al., J. Neurochem, 58, 1987-96 (1992).Somatostatin analogs typically, but need not, retain two importantmolecular features of somatostatin: its cyclic form and the 4 aminoacids involved in the binding to the somatostatin receptor (i.e., aminoacids 7-10 of the somatostatin sequence). A number of radiolabeledsomatostatin analogs (e.g., [¹¹¹In-DTPA-DPhe¹]octreotide) have beendeveloped that can be used to image these tumors using somatostatinreceptor scintigraphy. Krenning et al., Eur. J. Nucl. Med., 20, 716-731(1993). Somatostatin receptor scintigraphy is the most sensitive methodto localize the primary and metastatic disease in subjects with allpancreatic endocrine tumors and carcinoids. Gibril et al., Ann. Intern.Med. 125, 26-34 (1996). The localization of these tumors by somatostatinreceptor scintigraphy is due to the interaction of the radiolabeledanalogs with specific cell surface somatostatin receptors.

Multiple subtypes of somatostatin receptors are known, and almost allneuroendocrine tumors (carcinoids, pancreatic endocrine tumors) possessat least one subtype, frequently multiple subtypes. Somatostatinreceptor subtypes (sst₁, sst₂, sst₃, sst₄, and sst₅) have been isolatedand cloned. Both octreotide and lanreotide have high affinity forsomatostatin receptor sybtypes sst₂ and sst₃, lower affinity for sst₃and very low affinity for sst₁ and sst₄. Patel, Front. Neuroendocrinol.20, 157-198 (1999). Radiolabeled analogs of octreotide are rapidlyinternalized and the radiolabeled peptides can remain present in thecells for prolonged periods. Holland et al., Proc Assoc Am Physicians.111:63-69 (1999).

Radiotherapy using high doses of [¹¹¹In-DTPA-D-Phe¹]octreotide (DTPA:diethylenetriaminepentacetic acid), which emits auger and conversionelectrons, as well as ⁹⁰yttrium-labeled somatostatin analogs coupled bya DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)chelator, which can emit β-particles and give high radiation doses ofgreater penetrance, have been reported to inhibit tumor growth in bothanimal studies and in preliminary human studies. deJong et al., Q. J.Nucl. Med. 43, 356-366 (1999). Additional examples of somatostatinanalogs that are being evaluated for use in the radionuclide therapy oftumors include [DOTA⁰, Tyr³] octreotide (DOTATOC) labeled with ¹³¹I, ⁹⁰Yor ¹⁷⁷Lu. Peptide receptor radionuclide therapy (PRRT) usingradiolabeled DOTATOC has led to tumor responses in the majority ofsubjects, but has also posed problems with regard to renal andhematological toxicity. Reubi, Endocr, Rev. 24, 389-427 (2003). Anothersynthetic somatostatin-receptor targeting analog, [DOTA⁰, Tyr³]octreotate (DOTATATE) labeled with ¹⁷⁷Lu has recently been investigatedfor PRRT. J, Nucl. Med. 2005 Jan;46 Suppl 1;107S-14S; J Nucl Med. 2005Jan;46 Suppl 1:83S-91S1 Endocr Relat Cancer. 2005 Dec;12(4):683-99.

Despite good imaging and diagnostic results with ¹¹¹In labeled [DTPA⁰]octreotide (Octreoscan®) in the last few years, there have been severalreports describing new somatostatin radioligands for studying sstexpression. Some like [DOTA⁰, Tyr³] octreotide (DOTATOC) labeled with¹³¹I, ⁹⁰Y and ¹⁷⁷Lu are also being evaluated for use in the radionuclidetherapy of tumors (7). The new Peptide Receptor Radionuclide Therapy(PRRT) using radiolabeled DOTATOC has led to tumor responses in themajority of patients, but has also posed problems with renal andhematological toxicities Reubi, Endocr. Rev. 24, 389-427 (2003). Inprevious studies, kidney failures have been reported after treatmentwith DOTATOC labeled to β⁻ particle emitter ⁹⁰Y(8-10). In previouslycompleted clinical studies, it was observed that 10% to 34% patients hadcomplete remission following ⁹⁰Y-DOTATOC treatment (11). The results ofthese studies illustrate the partial treatment potentials of this agentand the possible higher relapse rates that may occur in the future (12).The primary challenges that ⁹⁰Y or ¹⁷⁷Lu labeled DOTATOC faces are renaltoxicities and incomplete treatments, especially in radio-resistanttumors.

Recent studies indicate that the presence of somatostatin receptors onother more common non-endocrine tumors may also be used for tumorlocalization or treatment. Halmos et al., J Clin Endocrinol Metab., 85,3509-12 (2000). Increased densities of somatostatin receptors are foundin various tumors of the central nervous system (meningiomas,astrocytomas, gliomas), some malignant lymphoid tumors (Hodgkin'sdisease, non-Hodgkin's disease), and in some cancers of the prostate,breast, kidney, liver, and lung. Jensen et al, J. Clin. EndocrinolMetab., 85(10), 3507-8 (2000). Somatostatin analogs have been shown tohave antiproliferative effects on breast, gastric, colorectal, prostate,thyroid, and lung tumors, and cytotoxic somatostatin analogues have beenshown to inhibit growth of human breast cancer, prostate cancer, renalcell carcinomas, and human glioblastomas. Kath et al , Recent ResultsCancer Res. 153, 23-43 (2000); Froidevaux et al., Curr. Med. Chem. 7,971-994 (2000). The effect of chemotherapeutic agents on the expressionof somatostatin receptors has been investigated using pancreatic tumorcells. Fueger et al., J. Nucl. Med. 42(12), 1856-62 (2001).

Gemcitabine (2′,2′-difluoro-2-deoxycytidine; dFdC) is a pyrrolidineanalog that has shown activity in various solid tumors, includingnon-small cell lung cancer (NSCLC), small cell lung cancer, head andneck squamous cell cancer, germ cell tumors, lymphomas (cutaneous T-celland Hodgkins' disease), mesothelioma, and tumors of the bladder, breast,ovary, cervix, pancreas, and biliary tract, as well as some hematologicmalignancies. The compound was first reported by Lilly ResearchLaboratories, Eli Lilly and Co.; Indianapolis, Ind. Hertel et al. CancerRes, 50, 4417-4422 (1990). Gemcitabine is a deoxycytidine analog withstructural similarities to cytarabine (Ara-C).

Gemcitabine is metabolized miracellularly by nucleoside kinases to theactive diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleosidemetabolites. The cytotoxic effect of gemcitabine is generally attributedto the actions of diphosphate and the triphosphate nucleosides, whichlead to inhibition of DNA synthesis. Gemcitabine diphosphate (dFdCDP)inhibits ribonucleoside reductase, which is responsible for catalyzingthe reactions that generate the deoxynucleoside triphosphates for DNAsynthesis. Inhibition of this enzyme by the diphosphate nucleosidecauses a reduction in the concentration of the deoxynucleotides,including dCTP. Gemcitabine triphosphate (dFdCTP) competes with dCTP forincorporation into DNA. The reduction in the intracellular concentrationof dCTP (by the action of the diphosphate) further enhances theincorporation of gemcitabine triphosphate into DNA, a process referredto as self-potentiation. After the gemcitabine nucleotide isincorporated into DNA, only one additional nucleotide is added to thegrowing DNA strand. Further DNA synthesis is inhibited, as DNApolymerase epsilon is unable to remove the gemcitabine nucleotide andrepair the growing DNA strand, resulting in what is known as maskedchain termination. Gemcitabine induces an S-phase arrest in the cellcycle, and triggers apoptosis in both human leukemic cells and solidtumors. Tolis et al., Eur. J. Cancer; 35, 797-808 (1999). In addition toits cytotoxic effect, gemcitabine is a potent radiosensitizer.Gemcitabine has been investigated as a radiosensitizer for rodent andhuman tumor cells, including those found in pancreatic, non-small celllung, head and neck, colorectal breast, and cervical cancer. Pauwels etal., Oncologist 10(1), 34-51 (2005).

OBJECTS OF THE INVENTION

It is an object of the invention to provide novel compounds andpharmaceutical compositions for the treatment of cancer and precancerousconditions.

It is another object of the invention to provide methods for treatingprecancerous conditions or cancer using compounds according to thepresent invention.

It is an additional object of the invention to provide methods fortreating precancerous conditions or cancer using compounds which enhanceexpression of somatostatin receptors in cancer cells in combination withagents which bind to somatostatin receptors to deliver cytotoxic agents.

Any one of these and/or other objects of the invention may be readilygleaned from a review of the description of the invention which follows.

SUMMARY OF THE INVENTION

The present invention provides a therapy effective for treating asubject afflicted with a cancer or precancerous condition. According toa first embodiment, the therapy includes administration of aradiopharmaceutical composition such as a somatostatin analog labeledwith a high Linear-Energy-Transfer (LET) α-emitter. An example of theradiopharmaceutical composition is ²¹³Bi-DOTATOC, but may be anysomatostatin analog, preferably selected from octreotide, lanreotide andvapreotide which have been radiolabeled with a Linear-Energy-Transfer(LET) α-emitter, preferably using a chelating moiety. In this method,the radiolabeled somatostatin analog is administered to a patient inneed of treatment in an effective amount to reduce the likelihood that aprecancerous condition will develop into cancer, to inhibit the growthof cancer or tumor and/or shrink the cancer or tumor in the patient orreduce the likelihood of metastasis of the cancer and/or tumor in thepatient. Remission of cancer in the patient is an alternative result inthe present method.

According to another embodiment, the therapy is a combination therapyinvolving administering a first therapeutic agent that increasesexpression of somatostatin receptors, and a second therapeutic agentthat selectively binds to a somatostatin receptor on the cancer cell anddelivers a cytotoxic compound or moiety to the cancer cell. An exampleof the first therapeutic agent is gemcitabine or an active gemcitabinemetabolite. The second therapeutic agent may include a recognitionligand that targets the somatostatin receptor, and a cytotoxic compound.Administration of the second therapeutic causes a deleterious effect onthe cancererous or precancerous cell. An example of a second therapeuticagent is a radiolabeled somatostatic analog, such as ²¹³Bi-DOTATOC,among numerous others.

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. As used in the description of the invention andthe appended claims, the singular forms “a”, “an”, and “the” areinclusive of their plural forms, unless contraindicated by the contextsurrounding such.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides structural formulas for deoxycytidine (CdR), cytarabine(Ara-C), and gemcitabine (dFdC).

FIG. 2 illustrates the structural relationship between somatostatin andthree somatostatin analogs, as shown in Cancer Medicine, 6^(th) edition(Frei et al., eds., Hamilton, Canada, 2003).

FIG. 3 provides a bar graph showing the cytotoxic effects of ¹⁷⁷Lu andlabeled DOTA and DOTATOC on the somatostatin receptor expressing cellline Capan-2.

FIG. 4 provides a bar graph showing the cytotoxic effects of ¹⁷⁷Lu and²¹³Bi labeled DOTA and DOTATOC on the somatostatin receptor negativecell line A549.

FIG. 5 provides a bar graph showing the cytotoxic effects ofradionuclide labeled DOTA and DOTATOC in combination with gemcitabinepretreatment on the somatostatin receptor expressing cell line Capan-2.

FIG. 6 provides a bar graph showing the apoptic effects of ¹⁷⁷Lu and²¹³Bi labeled DOTA and DOTATOC on the somatostatin receptor expressingcell line Capan-2.

FIG. 7 provides a bar graph showing the apoptic effects of radionuclidelabeled DOTA and DOTATOC in combination with gemcitabine pretreatment onthe somatostatin receptor expressing cell line Capan-2

FIG. 8 provides a bar graph showing increased ¹⁷⁷Lu-DOTATOC binding togemcitabine pretreated Capan-2 cells.

FIG. 9 provides a bar graph showing the increased effect of¹⁷⁷Lu-DOTATOC on cell viability at 24 hours for gemcitabine pretreatedCapan-2 cells.

FIG. 10 provides a bar graph showing the increased effect of¹⁷⁷Lu-DOTATOC on cell viability at 48 hours for gemcitabine pretreatedCapan-2 cells.

FIG. 11 provides a bar graph showing the increased effect of¹⁷⁷Lu-DOTATOC on apoptosis at 72 hours for gemcitabine pretreatedCapan-2 cells.

FIG. 12 shows the direct decay pathway (2%) of ²¹³Bi by α-emission tothe (3.980 MeV) β-particle emitter ²⁰⁹TI.

FIGS. 13A-B: Graphical comparisons of Tumor and Non-Tumor Bearing Ratsat 1 hour post injection of ²¹³Bi-DOTATOC. Columns, means, Bare, SEM,white columns represent non-tumor bearing rats and black columnsrepresent tumor bearing rats. Significance of p<0.05 is represented by a(*). FIG. 13A. Graph represents the high uptake organs, showinglocalization in the pancreas and adrenals was significantly higher inthe non-tumor. FIG. 13B. Graph represents the low uptake organs, showinglocalizations were significantly higher in both the stomach and muscle.

FIGS. 14 is a graphical comparison of T4 values in serum taken fromdifferent treatment groups of ²¹³Bi-DOTATOC at 23 days. Columns, means,Bars, SEM. Significance of p<0.05 is represented by a (*). Graphrepresents the decreased T4 value in (nmol/liter) with the increase ofinjected activity.

FIG. 15 shows that unaffected animals had normal-appearing glomeruli (A,upper left) and convoluted tubules (C, lower left), and no inflammatorycells were seen in the interstitium (A upper left, C lower left).Animals with interstitial nephritis had normal-appearing glomeruli (B,upper right) and tubules (D, lower right), but there were smallaggregates of mononuclear inflammatory cells in the interstitium(arrows, upper and lower right). Kidney sections were stained withhematoxylin and eosin. Bar=50 μm.

FIG. 16 shows a graph of small volume tumor bearing Lewis rats givenDOTATOC alone or a total 12.6 MBq of ²¹³Bi-DOTATOC. Symbols, means,Bars, SEM, Solid line with the square symbol represents the control,dashed line with the triangle symbol represents 12.6 MBq ²¹³Bi-DOTATOC.Rats were treated for 3 consecutive days with DOTATOC or ²¹³Bi-DOTATOC.

FIG. 17 shows a graph of large tumor volume bearing Lewis rats givenhigh 22.2 MBq and low 13.0 MBq doses of ²¹³Bi-DOTATOC. Symbols, means,Bars, SEM, Solid line with square symbol represents 13 MBq²¹³Bi-DOTATOC, Dashed line with the triangle symbol represents 22.2 MBq²¹³Bi-DOTATOC. Rats were treated for 3 consecutive days with²¹³Bi-DOTATOC.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following terms shall be used to describe the present invention.

The term “patient” is used throughout the specification to describe ananimal, generally a mammal and preferably a human, to whom treatment,including prophylactic treatment, with the compositions according to thepresent invention is provided. For treatment of those infections,conditions or disease states which are specific for a specific animalsuch as a human patient, the term patient refers to that specificanimal. While the use of the present invention to treat humansrepresents a primary aspect of the invention, veterinary applicationsare also intended.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or composition which, in context, isused to produce or effect an intended result, whether that resultrelates to the treatment of a tumor including a carcinogenic tumor orother cancer or the treatment of a precancerous lesion or other cell(s)which express abnormal or foreign proteins or immunogens on a cellsurface, in certain aspects related to the coadministration of acompound according to the present invention with another anticanceragent, the present invention relates to the enhancement of theanti-cancer effect of the anti-cancer compound, in instances whereenhancement of the expression of somatostatin receptors represents anaspect of the present invention, the term effective refers to an amountof a compound which appreciably or substantially increases theexpression of somatostatin receptors in cancer cells. This term subsumesall other effective amount or effective concentration terms which areotherwise described in the present application. With respect to ananti-cancer effect, that effect may be one or more of inhibiting furthergrowth of tumor or cancer cells, reducing the likelihood or eliminatingmetastatsis or producing cell death in the tumor or cancer cells,resulting in a shrinkage of the tumor or a reduction in the number ofcancer cells or preventing the regrowth of a tumor or cancer after thepatient's tumor or cancer is in remission.

The term “cancer” is used throughout the specification to refer to thepathological process that results in the formation and growth of acancerous or malignant neoplasm, i.e., abnormal tissue that grows bycellular proliferation, often more rapidly than normal and continues togrow after the stimuli that initiated the new growth cease. Malignantneoplasms show partial or complete lack of structural organization andfunctional coordination with the normal tissue and most invadesurrounding tissues, metastasize to several sites, and are likely torecur after attempted removal and to cause the death of the patientunless adequately treated. As used herein, the term neoplasia is used todescribe all cancerous disease states and embraces or encompasses thepathological process associated with malignant hematogenous, ascitic andsolid tumors. Representative cancers include, for example, stomach,colon, rectal, liver, pancreatic, lung, breast, cervix uteri, corpusuteri, ovary, prostate, testis, bladder, renal, brain/CNS, head andneck, throat, Hodgkin's disease, non-Hodgkin's lymphoma, multiplemyeloma, leukemia, melanoma, acute lymphocytic leukemia, acutemyelogenous leukemia, Ewing's sarcoma, small cell lung cancer,choriocarcinoma, rhabdomyosarcoma, Wilms' tumor, neuroblastoma, hairycell leukemia, mouth/pharynx, oesophagus, larynx, kidney cancer andlymphoma, among others, which may be treated by one or more compoundsaccording to the present invention.

The term “tumor” is used to describe a malignant or benign growth ortumefacent.

The term “precancerous” refers to a state in which cells are growing inan uncontrolled manner and where that growth has yet to develop into acancerous growth.

The term “anti-cancer compound” or “anti-cancer agent” is used todescribe any compound (including its derivatives) which may be used totreat cancer. Anti-cancer agents as described hereunder are a subset ofcytotoxic agents which may be used in the present invention. Exemplaryanti-cancer compounds for use in the present invention for linking witha somatostatin ligand include anti-metabolite agents which are broadlycharacterized as antimetabolites, inhibitors of topoisomerase I and II,alkylating agents and microtubule inhibitors (e.g., taxol), as well astyrosine kinase inhibitors (e.g., surafenib), EGF kinase inhibitors(e.g., tarceva or erlotinib) and ABL kinase inhibitors (e.g. gleevec orimatinib). Anti-cancer compounds for use in the present inventioninclude, for example, Aldesleukin; Alemtuzumab; alitretinoin;aliopurinol; altretamine; amifostine; anastrozole; arsenic trioxide;Asparaginase; BCG Live; bexarotene capsules; bexarotene gel; bleomycin;busulfan intravenous; busulfan oral; calusterone; capecitabine;carboplatin; carmustine; carmustine with Polifeprosan 20 implant;celecoxib; chlorambucil; cisplatin; cladribine; cyclophosphamide;cytarabine; cytarabine liposomal; dacarbazine; dactinomycin; actinomycinD; Darbepoetin alfa; daunorubicin liposomal; daunorubicin, daunomycin;Denileukin diftitox, dexrazoxane; docetaxel; doxorubicin: doxorubicinliposomal; Dromostanolone propionate; Elliott's B Solution; epirubicin;Epoetin alfa estramustine; etoposide phosphate, etoposide (VP-16),exemestane, Filgrastim, floxuridine (intraarterial); fludarabine;fluorouracil (5-FU); fulvestrant; gemtuzumab ozogamicin; gleevec(imatinib); goserelin acetate; hydroxyurea; Ibritumomab Tiuxetan;idarubicin; ifosfamide; imatinib mesylate; Interferon alfa-2a;Interferon alfa-2b; irinotecan; letrozole; leucovorin; levamisole;lomustine (CCNU); meclorethamine (nitrogen mustard); megestrol acetate;melphalan (L-PAM); mercaptopurine (6-MP); mesna; methotrexate;methoxsalen; mitomycin C; mitotane; mitoxantrone; nandrolonephenpropionate; Nofetumomab; LOddC; Oprelvekin; oxaliplatin; paclitaxel;pamidronate; pegademase; Pegaspargase; Pegfilgrastim; pentostatin;pipobroman; plicamycin; mithramycin; porfirmer sodium; procarbazine;quinacrine; Rasburicase; Rituximab; Sargramostim; streptozocin;surafenib; talbuvidine (LDT); talc; tamoxifen; tarceva (erlotinib);temozolomide; teniposide (VM-26); testolactone; thioguamine (6-TG);thiotepa; topotecan; toremifene; Tositumomab; Trastuzumab; tretinoin(ATRA); Uracil Mustard; valrubicin; valtorcitabine (monoval LDC);vinblastine; vinorelbine; zoledronate; and mixtures thereof, amongothers. Note that one of ordinary skill in the art may readily link aiigand which hinds to a somatostatin receptor with an anti-cancer agentas described hereunder for purposes of treating cancer.

The term “coadministration” or “combination therapy” is used to describea therapy in which at least two active compounds in effective amountsare used to treat cancer or another disease state or condition asotherwise described herein at the same time. Although the termcoadministration preferably includes the administration of two activecompounds to the patient at the same time, it is not necessary that thecompounds be administered to the patient at the same time, althougheffective amounts of the individual compounds will be present in thepatient at the same time. Compounds according to the present inventionmay be administered with one or more anti-cancer agent, includingantimetabolites, alkylating agents, iopoisomerase I and topoisomerase IIinhibitors as well as microtubule inhibitors, among others. Anticancercompounds for use in the present invention include those describedabove, and mixtures thereof, among others. Coadministration of one ofthe present compounds with another anticancer agent as otherwisedescribed herein will often result in a synergistic enhancement of theanticancer activity of the other anticancer agent, an unexpected result.One or more of the present compounds may also be coadministered withanother bioactive agent (e.g., antiviral agent, antihyperproliterativedisease agent; agents which treat chronic inflammatory disease, amongothers or as otherwise described herein).

The term “somatostatin receptor” refers to 5 distinct somatostatinreceptors (SSTR1-SSTR5) in various target tissues. See, Brazeau, et al.,Science, 129,77-79 (1973); Epelbaum., Prog. Neurobiol., 27, 63-100(1986); Yamada, et al., Proc. Natl. Acad. Sci. USA, 89, 251-255 (1992);Corness, et al., FEBS Lett., 321, 279-284 (1993); and Yamada, et al.,Biochem. Biophys. REs. Commun., 195, 844-852 (1993). All 5 human SSTRsubtypes bind SST-24 and SST-28 with high affinity and belong to thesuperfamiiv of guanine nucleotide binding protein-coupled receptors, inthe present invention, the single term “somatostatin receptor” refers tothe panoply of somatostatin subtypes unless a particular tissue andexpression of a particular subtype in such tissue is referred to. In thecase of a particular tissue, the term somatostatin receptor refers tosomatostatin receptors which are found in that type of cancer tissue.

According to a first embodiment, the present invention provides aradiopharmaceutical composition and method for treating a subjectafflicted with a cancer or precancerous condition by administering of asomatostatin analog radiolabeled with a high Linear-Energy-Transfer(LET) α-emitter. Examples of high LET α-emitters include ²¹¹At, ²¹³Bi,¹⁷⁷lutetium, and ¹¹¹indium. In preferred aspects of the invention, thesomatostatin analog is octreotide, lanreotide or vapreotide, which ischelated to any one of ²¹¹At, ²¹³Bi, ¹⁷⁷lutetium, and ¹¹¹indium,preferably ²¹³Bi. This compound may be administered along or incombination (coadministration) with another anticancer agent.

²¹³Bi decays mainly (98%) by β⁻-emission, with a 440-keV γ-emission anda half life (t_(1/2)) of 45.6 minutes to the ultra-short-livedhigh-energy (8.375-MeV) α-emitter ²¹³Po (t_(1/2)=4.2 microseconds).²¹³Bi also has a direct decay pathway (2%) by α-emission to the (3.980MeV) β⁻-particle emitter ²⁰⁹Tl (16). Accordingly, in one embodiment, thepresent invention provides a somatostatin analog [DOTA0 Tyr3] octreotide(DOTATOC) labelled ²¹³Bi.

Radionuclides such as ²¹³Bi that emit alpha particles offerradiotherapeutic advantages as they emit much higher energy particlesthan most of the betas, and yet their ranges are typically two orders ofmagnitude lower. Alpha particles have a high LET that is about 100 timesgreater than the beta particles, manifested by a higher RBE and a muchshorter range. Consequently, a much greater fraction of total energy isimparted to the targeted cancer cell and thus very few nuclear hits arerequired to kill the cell (24, 25, 26).

An exemplary method of preparation will now be described. ²¹³Bi can bereadily obtained from an “in-honse” ²²¹Ac/²¹³Bi radionuclide generatorsystem (National institutes of Health, National Cancer Institute,Bethesda, Md.). Prior to each elution, the ²²⁵Ac generator column wasfirst rinsed with distilled water and then flushed with air to removethe water, in order to selectively elute the²¹³Bi daughter, the columnwas eluted with 10 milliliter of 0.1 M hydrochloric acid. The eluate wasdiluted with water at 5.6 times the eluate volume of water (56milliliter). This dilution was loaded onto a MP-50 cation-exchangecolumn. This column was then reverse eluted with an additional 0.4milliliter of freshly prepared 0.1 M hydroiodic acid that contained thedesired ²¹³Bi.

Freshly eluted ²¹³Bismuth (4 MBq) was added to 0.5 μg of DOTATOCsolution and incubated for 5 minutes at 100° C. in a hot block. Prior toheating, the pH of the final solution was adjusted to 6 to 7 using 3 MNH₄OAc solution.

Incorporation yield (IC) was assessed using Silica Gel instantaneousthin layer chromatography (ITLC) with 0.9% sodium chloride as the mobilephase. The radiolabeled samples were diluted with 4 mMdiethylenetriamine pentaacetic acid (DTFA) at pH=4.1. Five microliter ofthe diluted sample was spotted on an ITLC silica gel strip and allowedto develop in a chromatographic chamber. Upon completion of themigration to the solvent front, the ITLC sample strips were allowed todry, cut in half and counted on a Wallac Wizard gamma counter (PerkinElmer, Boston, Mass.) to determine the IC. Radiochemical purity (RCP)was assessed via high performance liquid chromatography (HPLC) analysis.The liquid chromatography system (Thermo Separation Products, San Jose,Calif.) consisted of a multisolvent-delivery pump, an auto sampler, aradiometric detector (γ-RAM, IN/US Systems, Inc., Tampa, Fla.), and aC₁₈ 5 μm, 4.6×250 mm, reverse-phase HPLC column. The mobile phaseconsisted of Buffer A: 0.5 M Ammonium Acetate in HPLC grade water, pH5.5 and Buffer B: 100% HPLC grade methanol. The HPLC samples wereanalyzed with a 1:10 dilution in 4 mM DTPA. The flow rate was 1.0milliliter per minute and the retention time for the radiolabeledproduct was 14.0 to 14.5 minutes.

The radiolabeled product, ²¹³Bi-DOTATOC, was incubated at 37° C. in aCO₂ incubator for 1 hour in rat serum obtained from a male Lewis rat tostudy in vitro stability. After incubation the product was analyzed bythe ITLC and HPLC methods previously described. The radiolabelingincorporation yields and radiochemical purity by ITLC and HPLCG weregreater than 99.9% and greater than 95%, respectively. ²¹³Bi-DOTATOCdemonstrated acceptable stability and was unchanged after 1 hour of invitro incubation in rat serum. The biodistribution data demonstratedspecific binding to sst expressing tissues.

As will be described in greater detail below, it should be appreciatedthat the high LET α-emitter radiolabeled somastatin analog of thepresent invention may be administered to a patient using any knowntechnique.

According to still another embodiment, the present invention furtherprovides a pharmaceutical composition and method for treating asubjected afflicted with a cancer or precancerous condition byadministering a therapeutic agent comprising ²¹³Bi and apharmaceutically acceptable carrier.

The therapeutic agent may further include a targeting moiety thattargets the therapeutic agent to a selected mammalian cell. Those ofskill in the art will be familiar with suitable targeting moieties andwill be aware that the specific targeting moiety used will be dependantupon various factors including, for example, the mammalian cell that isselected. Accordingly, the targeting moiety may be a ligand or ligandanalog that is configured to bind to a receptor that is expressed orpreferentially expressed on the selected mammalian cell. Furthermore,the targeting moiety may be configured to facilitate internalization ofthe therapeutic agent by the cell.

Accordingly, the therapeutic agent may include a peptide, peptideanalog, peptide derivative, or a peptidomimetic compound. Thetherapeutic agent may further be effective in peptide receptorradionuclide therapy (PRRT).

Accordingly, where the selected cell is a cancer cell, the targetingmoiety maybe a somatostatin peptide, analog, or derivative thereof.Alternatively, the targeting moiety may include an octreotide or analogor derivative thereof, including vapreotide or lanreotide.

According to another embodiment, the present invention further providesa method of treating a subject afflicted with a cancer or precancerouscondition by increasing the expression level of somatostatin receptorsin the cancer cells and administering a therapeutic agent that binds tothe somatostatin receptors of the cancer. The expression level ofsomatostatin receptors in the cancer cells of the subject is optionallyincreased by administering a first therapeutic agent that increasessomotostatin receptor expression in the cancer cells. Administration ofa second therapeutic agent that binds to somatostatin receptors of thecancer provides a cytotoxic compound that has a deleterious effect onthe cancer, increasing the level of somatostatin receptors on the cancerof the subject provides advantages such as, for example, facilitatingassociation of the second therapeutic agent with the cancer, andpotentially decreasing the amount of the second therapeutic agentnecessary to provide the desired level of antitumor activity.

First Therapeutic Agent

According to an embodiment, a first therapeutic agent is administered tothe subject in order to increase the expression level of somotostatinreceptor in the cancer cells. Somatostatin receptors are proteins withan affinity for the hormone somatostatin, and include at least fivedifferent receptor subtypes. The first therapeutic agent may beeffective to increase the expression level of a particular somatostatinreceptor subtype (e.g., sst₁-sst₅) or it may increase the expression ofa plurality of somatostatin receptor subtypes. The increase inexpression of somatostatin receptors may occur immediately upon exposureto the first therapeutic agent, or it may occur after a certain periodof time. For example, somatostatin receptor expression levels mayincrease about 1,2, 3, or 4 days after initial exposure to the firsttherapeutic agent.

An increase in expression level of somatostatin receptors in the cancercells can be evidenced by an increase in the number of somatostatinreceptors in the cancer cells and/or the affinity of the somatostatinreceptors for their ligand, compared to the number and/or affinity ofsomatostatin receptors found in the cancer in the absence ofadministration of the first therapeutic agent. Preferably, thesomatostatin receptors are expressed on the surface of the cancer cellswhere they may readily bind the second therapeutic agent. An increase inexpression level of somatostatin receptors may include stimulation ofexpression of somatostatin receptors on cancer ceils that previously didnot express somatostatin receptors, in addition to increased expressionof somatostatin receptors by cells that previously expressedsomatostatin receptors at a lower level. In an additional aspect of theinvention, somatostatin receptor expression is increased to a higherextent in cancer cells relative to normal, non-neoplastic tissue.

The first therapeutic agent may increase the expression level ofsomatostain receptors in the cancer in any of a variety of ways. Forinstance, in one embodiment of the invention, the first therapeuticagent may increase the expression level of somatostatin receptors in thecancer by inducing cell cycle arrest. Cell cycle arrest may occur duringany of the cell cycle phases. For example, cell cycle arrest may occurin the G0/G1 phase, the G2/M phase, or the S phase. While not intendingto be bound by theory, cell cycle arrest may lead to increasedsomatostatin receptor expression by retaining the cell in a phase inwhich somatostatin receptor is expressed. For example, somatostatinreceptor expression may occur during the S phase in order to regulatecell growth. Cell cycle arrest in S phase may lead to an increase insomatostatin receptor expression. Cell cycle arrest may occur as theresult of a variety of different effects. For instance, cell cyclearrest may occur as a result of the inhibition of DNA synthesis. DMAsynthesis may be inhibited, for example, due to chain temrination byincorporation of an altered nucleoside analog (e.g., dFdCTP), and/or byinhibition of an enzyme involved in DNA synthesis such as ribonucleotidereductase, which is a rate-limiting enzyme m DNA synthesis (e.g., viathe activity of dFdCDP).

Accordingly, in some embodiments of the invention, the first therapeuticagent may be a DNA synthesis inhibiting agent. A wide variety ofantitumor agents are known to those skilled in the art that inhibit DNAsynthesis. For example, one class of DNA synthesis inhibiting agents arealkylating agents (e.g., classes of alkylating agents such as nitrogenmustards, aziridines, epoxides, nitrosoureas, triaztnes, andhydrazines).

According to one embodiment of the invention, the first therapeuticagent is a DNA synthesis inhibiting agent which is a nucleoside analog(e.g., a purine or pyrimidine analog). Nucleoside analogs includecompounds wherein, for example, the sugar or base is chemicallymodified. Many “analogous” forms of purines and pyrimidines are known inthe art, an many of them are in use as chemotherapeutic agents. Purineanalogs include, for example, mercaptopurine, azathioprine, thioguanine,deoxocoformycin, fludaribine, cladribine, and hydroxyurea.

Pyrimidine is a nitrogen-containing, six-membered ring that is bonded tothe C-1 position of ribose to form a pyrimidine nucleoside. Pyrimidinenucleotides in DNA include cytosine and thymine. Embodiments of theinvention include analogs wherein a deoxyribose sugar of the pyrimidineanalog includes halogen substituents at the 2-deoxy portion of thesugar. The halogen used may be fluorine, chlorine, bromine, or iodine.For example, in one embodiment of the invention, the pyrimidine analogincludes fluorine substituents at the 2-deoxy position (e.g.,2′,2′-difluoro-2′-deoxycytidine (gemcitabine)). Pyrimidine analogsinclude, for example, 5-fluorouracil, cytosine arabinoside,5-azacytidine, and gemcitabine.

Typically, purine or pyrimidine analogs are active only after metabolicconversion to the active nucleotide form. These nucleotide analogs maythus not only may be incorporated into DNA but also can mimic thenatural effector compounds in regulatory pathways.

An exemplary but not exhaustive list of nucleoside analogs can be foundin U.S. Pat. No. 6,989,452 and includes 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N⁶-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, □-D-mannosylqueosine,5-metboxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic add (v), pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. In addition, the conventional bases may be replacedby halogenated bases. Furthermore, the 2′-furanose position on the basecan have a non-charged bulky group substitution. Examples of non-chargedbulky groups include branched alkyls, sugars and branched sugars. Seealso U.S. Pat. No. 6,077,668 for additional nucleoside and nucleotideanalogs.

The DNA synthesis inhibiting agent may include a class of compounds thattarget a particular portion of DNA synthesis. For example, the DNAsynthesis inhibiting agent may be a ribonucleotide reductase inhibitor.Examples of agents that may be used to inhibit ribonucleotide reductaseinclude hydroxyurea, guanazole, gemcitabine, fludarabine, andthiosemicarbazone derivatives. Examples of suitable ribonucleotidereductase inhibitors are described by Szekeres et al. (Szekeres et al.,Crit. Rev. Clin. Lab. Sci., 34, 503 (199)).

The first therapeutic agent, in some embodiments of the invention, alsoincludes active metabolites of the first therapeutic agent. For example,pyriraidine nucleoside analogs are typically activated by metabolic conversion to the active nucleotide, which may be a monophosphate,diphosphate, or triphosphate. Active metabolites, as defined herein, aremetabolically formed analogs of an agent that play a role m the activityof the agent. The active metabolites of pyrimidine nucleoside analogsinclude phosphate derivatives of pyrimidine nucleoside analogs. Forexample, gemcitabine is metabolized intracellularly by nucleosidekinases to the active diphosphate and triphosphate nucleosidemetabolites, and thus gemcitabine diphosphate and gemcitabinetriphosphate are active metabolites included in embodiments of theinvention. These gemcitabine metabolites play a role in the activity ofgemcitabine through inhibition of ribonucleoside reductase (bygemcitabine diphosphate) and competition with dCTP for incorporationinto DNA (by gemcitabine triphosphate).

Second Therapeutic Agent

The present invention may further include administration of a secondtherapeutic agent that binds to somatostatin receptors of the cancer.For instance, pre-treatment with a first therapeutic agent (e.g.,gemcitabine) may be followed by administration of a second therapeuticagent, such as, but not necessarily limited to, the radiopharameuticalcompositions described above. The second therapeutic agent may include arecognition ligand that binds to a somatostatin receptor, but does notsignificantly bind to other cell surface components. Binding of therecognition ligand to a somatostatin receptor may bring the secondtherapeutic agent into proximity with a cancer cell. The secondtherapeutic agent may further include a cytotoxic agent that has adeleterious effect on the cancer when the second therapeutic agent isbrought into proximity with a cancer cell. In addition to bringing thecytotoxic agent into proximity with the cancer cell, interaction of thesecond therapeutic agent with the somatostatin receptor may facilitateuptake of the second therapeutic agent into the cell. Hofland et al., J.Nucl. Med. 46, Suppl. 1, 191S-8S (2005).

The second therapeutic agent may include a recognition ligand thatselectively binds to somatostain receptors. A recognition ligand that“selectively binds” a somatostatin receptor is one that will, underappropriate (e.g., physiological) conditions, interact with asomatostatin receptor preferentially compared to other cell surfacecomponents, such as non-somatostatin receptors. Recognition ligandsinclude somatostatin analogs, antibodies, and other types of proteins,peptides, small organic molecules, and the like that selectively bind tosomatostatin receptors. Recognition ligands may bind to all subtypes ofsomatostatin receptors, or they may selectively bind to one or moresomatostatin receptor subtypes. For example, embodiments of theinvention include recognition ligands that selectively bind tosomatostatin receptor subtype sst₂. Examples of recognition ligands thatbind to specific somatostatin receptor subtypes are described by Reubiet al, (Reubi et al., Eur. J. Pharmacol., 5, 45-9 (2002).

Antibodies

The recognition ligand may take the form of an antibody that selectivelybinds to somatostatin receptors. Antibodies, as defined herein, includevertebrate antibodies, hybrid antibodies, chimeric antibodies, humanizedantibodies, altered antibodies, univalent antibodies, monoclonal andpolyclonal antibodies, Fab proteins, scFv single chain domain, scFvdimers of single chain domain or diabodies, minibodies, bi-specificminibodies, and aggregates of targeting domains. Monoclonal andpolyclonal anti-somatostatin receptor antibodies are well known to theart. For example, commercially available polyclonal antibodies that arederived from rabbit, and having specific epitopes for SSTr subtype 1, 2,4, and others are available from Abcam Inc., Cambridge, Mass.

Somatostatin Analogs

Alternatively, or additionally, the recognition ligane may take the formof somatostatin or a somatostatin analog. Somatostatin is generallyexpressed as a tetradecapeptide, and is the natural recognition ligandfor somatostatin receptors. The tetradecapeptide form of somatostatinhas the amino acid sequence ofAla-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO:1).While somatostatin itself may be used as a recognition ligand, syntheticsomatostatin analogs, which incorporate a Phe-D-Trp-Lys-Thr (or similarsequence) and which are metabolically stabilized at both the N- andC-terminals, have been developed for clinical applications. For example,three commercially available somatostatin analogs (i.e. octreotide[D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH); OCT (Rosenberg et al., CancerJ. Surg. 34, 223-229 (1991)], lanreotide[D-□Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2; LAN (Giusti et al., Eur. J.Clin. Invest. 27, 277-284 (1997))] and vapreotide[D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2; VAP (Stiefel et al., SupportCare Cancer 1:57-58 (1993))]) have been shown to be effective incontrolling the growth of some human tumors. These SST analogs all havesimilar binding profiles for four of the five human sst subtypes (i.e. ahigh affinity for human sst2 and sst5, moderate affinity for human sst3,and very low affinity for human sst1), but LAN and VAP have a moderateaffinity for human sst4, whereas OCT has little or no affinity for thishuman sst (for review, see Lamberts et al., N. Engl. J. Med. 334,246-254 (1996).

In one embodiment, a somatostatin analog includes amino acids 7-10, orderivatives or analogs thereof such as D-amino acids, of somatostatin,since this sequence is thought to be important for providing affinityfor the somatostatin receptor. See Veber et al., Nature 292, 55-8(1981). For example, Veber et al. describe the preparation of a numberof cyclic hexapeptide somatostatin analogs in which nine ofsomatostatin's amino acids are replaced with a single proline aminoacid, while Bauer et al., describe the preparation of a series ofoctapeptide cysteine-bridged analogs of somatostatin (Bauer et al., LifeSci, 31, 1133-40 (1982)). FIG. 2 illustrates the structural relationshipbetween somatostatin, the Veber hexapeptide, and the octapeptide analogsoctreotide and vapreotide. Accordingly, some embodiments of theinvention include the use of somatostatin analogs that include 5-10amino acids, or more preferably 6-8 amino acids. According to someembodiments, these somatostatin analogs include the binding region aminoacids from positions 7 to 10 of somatostatin.

Examples of octapeptide somatostatin analogs suitable for use asrecognition ligands in embodiments of the invention include octreotide(D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH), lanreotide(D-βNal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH₂) and vapreotide(D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH₂). Many additional somatostatinanalogs have been synthesized that are suitable for use as recognitionligands. See, Schally A V. Anticancer Drugs 5, 115-30 (1994); Cai etal., Proc. Natl. Acad. Sci. U S A, 83, 1896-900 (1986); Shimon, I.,Endocrine, 20, 265-9 (2003). See also U.S. Pat. Nos, 6,051,554;6,214,316: 5,932,189; 6,358,491; 6,316,414; and 6,930,088, relevantportions of which are incorporated herein. While somatostatin analogsare typically peptides, non-peptide somatostatin analogs have also beendeveloped and may be used in embodiments of the invention. Non-peptidesomatostatin analogs include somatostatin peptidomimetics, which arecompounds containing non-peptidic structural elements that are capableof mimicking or antagonizing the biological action of naturalsomatostatin. Peptidomimetics typically lack classic peptidecharacteristics such as, for example, enzymatically scissile peptidicbonds. See the IUPAC definition of a peptidomimetic compound. Wermuth etal., Pure and Applied Chemistry, 70, 1129-1143 (1998).

As stated above, the second therapeutic agent may also include acytotoxic compound. For the purposes of the present disclosure, acytotoxic compound is one that has a deleterious effect on a cell, suchas causing cell death, inhibition of cell growth and/or interfering withthe cell's ability to divide. The cytotoxic compound is brought intoproximity of the cancer cells by use of the recognition ligand, asdescribed above, and has a deleterious effect on proximal cancer cells.The deleterious effect may include, for example, direct cytotoxicity,cytostasis, or apoptosis. A wide variety of cytotoxic agents areavailable and known to those skilled in the art. A cytotoxic agent is asubstance that is potentially genotoxic, oncogenic, mutagenic,teratogenic or in any way hazardous to a cell. Cytotoxic agents mayinclude, for example, antitumor agents, toxic agents such as ricin, andradionuclides. Other examples include bacterial toxins (e.g.,Pseudomonas exotoxin), ricin A-chain, daunorubicin, doxorubicin,2-pyrrolinodoxorubucin, methotrexate, and ribosome inhibitors (e.g.,trichosantin). An example of a suitable second therapeutic agent is acytotoxic agent such as doxorubicin or 2-pyrrolinodoxorubucin linked toan octapeptide somatostatin analog. See Nagy et al., Proc. Natl. Acad.Sci. U S A 95, 1794-9 (1998). Additional cytotoxic somatostatin analogsare described by Hofland et al. (Hofland et al., J. Nucl. Med. 46,Suppl. 1, 191S-8S (2005). Other cytotoxic agents (anticancer) agentshave been discussed hereinabove. One or more of these traditionalcytotoxic agent may be modified so that they are linked to thesomatostatin recognition ligand, either covalently or through chelation.Alternatively, these anticancer agents may be coadministered with thefirst therapeutic compound and the second therapeutic compound tofurther treat cancer according to the present invention.

The cytotoxic compound included in the second therapeutic agent may be aradionuclide. Radionuclides provide a deleterious effect on cancer cellsthrough release of high energy particles such as α-, β-, andγ-particles. A wide variety of radionuclides suitable for use as acytotoxic compound are known to those skilled in that art. Examplesinclude ⁶⁷gallium, ⁶⁸gallium, ⁷¹arsenic, ⁷²arsenic, ⁶⁵zinc, ⁷⁶bromine,²⁰¹thallium, ^(99m)technicium, ⁴⁸vanadium, and ⁴⁹vanadium, as well asradionuclides more typically used in therapeutic applications such as⁹⁰yttrium, ¹¹¹indium, ¹⁷⁷lutetium, ²²⁵actinium, ²⁰⁹bismuth, ²¹²bismuth,²¹³bismuth, ⁶⁴copper, ⁶⁷copper, ⁷⁶arsenic, ⁷⁷arsenic, ²⁰³lead, ²⁰⁹lead,²¹²lead, ¹⁶⁶holmium, ¹⁵³promethium, ¹⁸⁶rhenium, ¹⁸⁸rhenium, and²¹¹astatine.

Radionuclides that release high-linear energy transfer (LET) α-particlesmay be used and are preferable, as these particles are highly toxic buthave a relatively short range (e.g., two orders of magnitude lower thanβ-particles) and are thus less likely to damage non-proximal tissue.Examples of radionuclides suitable for use as the cytotoxic compoundinclude ²¹³bismuth, ¹⁷⁷lutetium, and ¹¹¹indium.

In addition to cytotoxic agents which are inherently toxic, cytotoxicagents which require external activation to become cytotoxic may also beused. These include cytotoxic agents that are chemically, enzymatically,or electromagnetically activated. For example, somatostatin analogsincluding an attached superparamagnetic nanoparticle could be used,wherein the superparamagnetic nanoparticle becomes cytotoxic uponexposure of the nanoparticle to electromagnetic radiation, e.g., causingthermal ablation.

The cytotoxic compound and the recognition ligand of the secondtherapeutic agent may be associated together in any of a variety ofways. The association between the cytotoxic compound and the recognitionligand can be covalent or non-covalent. For example, the cytotoxiccompound and the recognition ligand may be non-covalently associated byimbedding them together in a structure such as a liposome.Alternatively, the cytotoxic compound may be covalently bound to arecognition ligand through reaction between the cytotoxic agent and therecognition ligand. For example, to covalently bind somatostatin analogoctapeptides to doxorubicin, conjugation was performed by coupling usingN-9-fluorenylmethoxycarbonyl (N-Fmoc). Nagy et al., Proc. Natl. Acad.Sci. U S A., 17, 1794-9 (1998). Other coupling agents that may be usedinclude dicyclohexyl carbodiimide and n-hydroxy succinamide.

Chelating agents that bind to the cytotoxic compound can be used toassociate the cyototoxic agents to a recognition ligand. Use ofchelating agents is often preferred when associating inorganic compoundssuch as radionuclides with recognition ligands. A variety of chelatingagents are a vailable that may be used to associate the cytotoxiccompound to the recognition ligand. Examples of bifunctional chelatingagents (i.e., chelating agents that include an array of metal-bindinggroups plus a moiety capable of covalent binding to a protein substrate)include chelating proteins, diethylenetriamine-pentaacetic acid (DTPA),imino-diacetic acid (IDA), nitrilo-triacetic acid (NTA),ethylenediamine-tetraacetic acid (EDTA), diaminocyclohexame-tetraaceticacid (DCTA), porphyrin, deferoxamine,tetraagacyclo-tetradecane-tetraacetate (TETA), and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). It isto be understood that the invention is not intended to be limited to aparticular chelating agent used. Rather, one of skill in the art canselect a chelating agent based on the compound, such as the metal orradionuclide, to be incorporated and the clinical objectives as well asthe compatability of the chelating agent with the chemistry associatedwith the ligand. Examplary classes of chelates include open-chainpolyaminocarboxylates, such as EDTA (ethylenediaminetetraacetic acid)and DTPA (diethylenetriaminepentaacetate); AZA macrocyclics such ascyclen (1,4,7,10-tetraazacyclotradecane), cylamd(1,4,8,11-tetraazacyclotetradecane), bridged-cyclam(1,4,8,11-tetraazabicyclo[6.6.]hexadecane), et-cyclam(1,4-ethano-1,4,8,11-tetraazacyclotetradecane), cylamdione(3,9-dioxy-1,4,8,11-tetraazacyclotetradecane), and diamsar(1,8-diamine-3,6,10,13,16,19-hexaazabicyclo(6,6,6)eicosane);polyaminocarboxylic macrocycles such as DOTA:(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TRITA:(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid), TETA;(trimethylenetetramine), bridged-cyclam-2a;(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-1,8-di(methanephosphonicacid)), DO3A:(1,4,7-tris(carboxymethyl)-1,4,7,10-tetraaxaeyclododecane), DO2A:(1,4,7,10-tetraazacyclododecane-1,7-bis(acetic acid)); andpolyaminophosphonate macrocycles such as DOTP:(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)),DO3P: (1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonicacid)), and DO2P:(1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid)).

An example illustrating the structure and function of a chelating agentis provided by DTPA, which includes a backbone of three nitrogen atomslinked by two ethylene chains. Extending from the nitrogen atoms on thebackbone are five carboxymethyl moieties. The carboxymethyl groups maybe reacted using conventional peptide chemistry to form an amide bondwith an amino acid residue present on an antibody or other proteinmolecule. The other four carboxymethyl moieties, together with the threenitrogen atoms, then remain available for metal binding. See, forexample, U.S. Pat. No. 5,057,302.

DOTA is a particularly suitable chelating agent for use with many metalion radionuclides such as bismuth, lutetium, and indium. DOTA may besynthesized by alkylating cyclers with chloroacetic acid or bromoaceticacid, and forms especially stable complexes with lanthanides, which areretained by the chelating agent with very high kinetic stability. DOTAmay be readily bound to a recognition ligand using, for example, theDOTA-tris(tert-butyl ester). Examples of recognition ligands coupled toradionuclides using DOTA, for use as second therapeutic agents, include¹⁷⁷lutetium coupled to octreotide (¹⁷⁷Lu-DOTATOC) and ²¹³bismuth coupledto octreotide (²¹³Bi-DOTATOC), as described further in the examplesherein.

While not intending to be bound by theory, in addition to its functionalrole in coupling a cytotoxic compound to a recognition ligand, chelatingagents may also piav a role in the antitumor activity of the secondtherapeutic agent by providing resistance to degradation or byfunctioning as a scavenger subsequent to uptake by the cancer cell.

A particularly preferred second therapeutic agent is described in U.S.provisional patent application U.S. Ser. No. 60/703,810, filed Jul. 29,2005, entitled “Radioisotopic Therapeutic Agent and Method” and inAppendix A hereto.

Antitumor Therapy

According to some embodiments, the present invention provides a methodof treating a subject afflicted with a cancer or precancerous conditionby administering a therapeutic agent that binds to the somatostatinreceptors of the cancer cell and delivers a cytotoxic agent that has adeleterious effect on the cancer. The method may further compriseincreasing the expression level of somatostatin receptors in the cancercell prior to administering the cytotoxic agent.

The cancer that is treated using the present methods may be primarycancer or it may be metastatic cancer. The present methods are suitablefor the treatment of any cancer or precancerous condition in whichsomatostatin receptors are expressed by the cancer cells. Somatostatinreceptors are generally expressed in higher levels by cancer cellsrelative to the cells of normal tissue. Preferably, the cancer cellsbear a greater number of somatostatin receptors than are found innon-cancerous tissue. Somatostatin receptors have been demonstrated onthe surface of a wide variety of cancer cells. In particular,somatostatin receptors have been demonstrated in cancers that includecancer cells with amine precursor uptake and decarboxylation properties.Furthermore, the present methods axe particularly suitable for thetreatment of any cancer or precancerous condition in which theexpression level of somatostatin receptors in the cancer cells can beincreased.

The invention is particularly well-suited for treatment of cancertumors. Accordingly, in one embodiment, the present invention provides amethod for treating a subject that is afflicted with a cancerous orprecancerous tumor, such as a catcinoma, a sarcoma, or a lymphoma.Preferably, the cancer treated is one that has neural and/or hormonalresponsiveness. Typically these cancers are referred to as “-omas.” Inan embodiment, the cancer treated is one that is treatable withgemcitabine either “on label” or “off label.” Examples of such cancersinclude pancreatic cancer, breast cancer, small cell lung cancer,pituitary adenomas and oilier neuroendocrine carcinomas.

As noted earlier, increased densities of somatostatin receptors arefound in various cancers of the central nervous system (meningiomas,astrocytomas, gliomas), some malignant lymphoid cancers (Hodgkin'sdisease, non-Hodgkin's disease), and in some cancers of the prostate,breast, kidney, liver, and lung, among others. Accordingly, embodimentsof the invention may be directed to treatment of somatostatin-receptorexpressing cancer cells. Cancers that have been found to expresssomatostatin receptors include a variety of different types of cancer,such as breast, pancreatic, gastric, prostate, renal, colorectal,thyroid, long, kidney, liver, central nervous system, and malignantlymphoid cancers. Somatostatin receptor expression can be demonstratedat the mRNA level using a variety of methods, such as in situhybridization, RNAse protection assays, reverse transcriptase polymerasechain reaction, or autoradiography. The presence of somatostatinreceptors can also be demonstrated using other methods such asimmunohistochemistry. In further embodiments of the invention, themethod is used to treat tumors resulting from cancers of neuroendocrineorigin such as somatotrophs tumors of the anterior pituitary andpancreatic islet-cell tumors.

Treating a subject may pro vide a reduction in tumor load or a decreasein tumor growth in a subject in response to administration of the firstand second therapeutic agent. The reduction in tumor load may representa direct decrease in tumor mass, or it may be measured in terms of tumorgrowth delay, which is calculated by subtracting the average time forcontrol tumors to grow over to a certain volume from the time requiredfor treated tumors to grow to the same volume. The subject is preferablya mammal, such as a domesticated farm animal (e.g., cow, horse, pig) orpet (e.g., dog, cat). More preferably, the subject is a human. Thetreatment may result in a decrease in the likelihood that precanceroustissue will develop into cancer, in addition, in preferred embodiments,the treatment may result, in decreasing the likelihood that metastasisof the cancer will occur, and optimally results in remission of thetreated cancer.

Administration and Formulation of Therapeutic Agents

Methods of administering small molecule therapeutic agents suchgemcitabine are well-known in the art. Dosage calculation for antitumoragents are exemplified, for example, by Guraev. Gumey H., J. Clin.Oncol, 14, 2590-2611. Methods for extrapolation of effective dosages inmice, and other animals, to humans are also known in the art; forexample, see U.S. Pat. No. 4,938,949. Dosage calculations for individualtherapeutic agents may also he readily determined from the literature bythose skilled in the art. For example, dosing and clinical studies ofsomatostatin analogs, gemcitabine, and numerous other drags may be foundat the U.S. Food and Drug Administration Center for Drug Evaluation andResearch website, and from literature that accompanies commerciallyavailable therapeutic agents, such as product literature for GEMZAR (EliLilly and Company), the commercially available injectable form ofgemcitabine HCL (PV 4046 AMP; Eli Lilly and Company, 2005).

For chemotherapeutic agents such as gemcitabine, dosages useful in thecombination therapy of the invention include any dosage which is knownto be useful or applicable for monotherapy using gemcitabine, or forother combination therapies that involve gemcitabine.

The therapeutic agents described in the present disclosure can beadministered to a subject alone or together (coadministered, optionally,but not necessarily, in a single formulation) with other active agentsas described herein, and are preferably administered with apharmaceutically acceptable buffer. The therapeutic agents can becombined with a variety of physiological acceptable carriers, additivesfor delivery to a subject, including a variety of diluents or excipientsknown to those of ordinary skill in the art. For example, for parenteraladministration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit acti vity of the peptide, can be used. Other suitablecarriers include, but are not limited to, alcohol, phosphate bufferedsaline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the therapeuticagent (i.e., the active agent) into association with a carrier thatconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing theactive agent into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations. The methods of the invention includeadministering the therapeutic agents to a subject in an amount effective to produce the desired effect. The therapeutic agents can beadministered as a single dose or in multiple doses. Useful dosages ofthe active agents can be determined by comparing their in vitro activityand the in vivo activity in animal models.

In the embodiment in which a first therapeutic agent is administered toincreasing receptor expression and a second therapeutic agent isadministered that targets the receptors, the first and secondtherapeutic agents may be administered together or separately in asingle dose or in multiple doses. Administration of the secondtherapeutic agent after administration of the first therapeutic agentprovides the advantage of providing time for the first therapeutic agentto enrich somatostatin receptor expression in the cancer cells, therebyfacilitating targeting of the second therapeutic agent to the cancer.The second therapeutic agent may be administered as much as two weeksafter the administration of the first therapeutic agent or as little astwo days afterward or even sooner, such as 24 hours after administrationof the first therapeutic agent. In a preferred embodiment, the secondtherapeutic is administered about 3 to 6 days following theadministration of the fust therapeutic agent.

Moreover, treatment of a subject afilicted with a cancer or precancerouscondition by administering a first and second therapeutic may result inan additive effect. More preferably, treatment by administering a firstand second therapeutic agent results in a synergistic therapeuticeffect. A synergistic effect, as defined herein, occurs when treatmentby a first therapeutic agent in conjunction with a second therapeuticagent results in a reduction in tumor load or growth delay that isgreater than the reduction in tumor load or growth delay that isobserved when the effects of separate treatment by the first therapeuticagent and the second therapeutic agent of the invention are addedtogether, where the dosages and treatment schedules are otherwise thesame when used individually or in combination. The comparison of thecombined treatment with the effects of separate treatment, addedtogether, result in a ratio that will be greater than 1 (i.e., greaterthan 100%) if a synergistic effect is present. Preferably, a synergisticeffect with a ratio of at least 2 (i.e., at least 200%) is provided bythe method of the invention, and more preferably the synergistic effecthas a ratio of at least 3 (i.e., at least 300%).

The therapeutic agents of the present invention are preferablyformulated in pharmaceutical compositions and then, in accordance withthe methods of the invention, administered to a subject, such as a humansubject, in a variety of forms adapted to the chosen route ofadministration. For example, the therapeutic agents may be formulatedfor intravenous administration. The formulations may, however, includethose suitable for oral, rectal, vaginal, topical, nasal, ophthalmic, orother parenteral administration (including subcutaneous, intramuscular,intraperitoneal and intratumoral, in addition to intravenous)administration.

Formulations suitable for parenteral administration conveniently includea sterile aqueous preparation of the active agent, or dispersions ofsterile powders of the active agent, which are preferably isotonic withthe blood of the recipient. Parenteral administration of the therapeuticagents (e.g., through an I.V. drip) is an additional form ofadministration. Isotonic agents that can be included in the liquidpreparation include sugars, butlers, and sodium chloride. Solutions ofthe active agents can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions of the active agent can be prepared inwater, ethanol, a polyol (such as glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, glycerol esters,and mixtures thereof. The ultimate dosage form is sterile, fluid, andstable under the conditions of manufacture and storage. The necessaryfluidity can be achieved, for example, by using liposomes, by employingthe appropriate particle size in the case of dispersions, or by usingsurfactants. Sterilization of a liquid preparation can be achieved byany convenient method that preserves the bioactivity of the activeagent, preferably by filter sterilization. Preferred methods forpreparing powders include vacuum drying and freeze drying of the sterileinjectable solutions. Subsequent microbial contamination can heprevented using various antimicrobial agents, for example,antibacterial, antiviral and antifungal agents including parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Absorptionof the active agents over a prolonged period can be achieved byincluding agents for delaying, for example, aluminum monostearate andgelatin.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder or granules, as liposomes containing thefirst and/or second therapeutic agents, or as a solution or suspensionin an aqueous liquor or non-aqueous liquid such as a syrup, an elixir,an emulsion, or a draught. Such compositions and preparations maycontain at least about 0.1 wt % of the active agent. The amounts of thetherapeutic agents should be such that the dosage level will beeffective to produce the desired result in the subject.

Nasal spray formulations include purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes. Formulations for rectal orvaginal administration may be presented as a suppository with a suitablecarrier such as cocoa butter, or hydrogenated fats or hydrogenated fattycarboxylic acids. Ophthalmic formulations are prepared by a similarmethod to the nasal spray, except that the pH and isotonic factors arepreferably adjusted to match that of the eye. Topical formulationsinclude the active agent dissolved or suspended in one or more mediasuch as mineral oil, petroleum, polyhydroxy alcohols, or other basesused for topical pharmaceutical formulations.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; an excipient such as diealcium phosphate; adisintegrating: agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, fructose, lactose, or aspartame; and a natural orartificial flavoring agent. When the unit dosage form is a capsule, itmay further contain a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present, as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac, sugar, and the like. A syrup or elixir may contain one ormore of a sweetening agent, a preservative such as methyl- orpropylparaben, an agent to retard crystallization of the sugar, an agentto increase the solubility of any other ingredient, such as a polyhydricalcohol, for example glycerol or sorbitol, a dye, and flavoring agent.The material used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The active agent may be incorporatedinto sustained-release preparations and devices.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scope ofthe invention as set forth herein.

EXAMPLES Single Radiolabeled Somatostatin Analog as Anti-Cancer AgentExample 1

Toxicology was assessed at 25 days in 4 groups of tumor bearing maleLewis rats (average volume 0.75±0.3 mm³). Rats in the first group,Cohort 1, were injected on day 1 only. Rats in second cohort wereinjected on days 1 and 2, while rats in the third cohort were injectedon days 1,2, and 3. Rats were injected with 2.56 μg, 0.5 μg, 0.5 μgDOTATOC, on day 1, day 2 and day 3, respectively, with a nominalactivity of 3.7 MBq. Each dose was divided into 2 injections at 1 hourintervals. The rats received the following cumulative average activitiesper group: Cohort 1 (N=3) received 4.3±0.7 MBq, Cohort 2 (N=3) received9.0±0.4 MBq, and Cohort 3 (N=4) received 12.6±0.3 MBq of ²¹³Bi-DOTATOCCohort 4 (N=4) was added as control group and injected twice daily withunlabelied DOTATOC on three consecutive days.

After 24 days, the animals were put into a metabolic cage to collecturine samples for creatinine clearance analysis. Creatine clearance wasdetermined as previously described (20). After 24 hour urine collection,the animals were euthanized with halothane. For blood collection, acardiac puncture was performed. Blood analysis consisted of hemoglobin(Hgb), hematocrit (Hct), red blood cells (RBCs), and white blood cells(WBC's) with differential, and platelets (Pfs). Additionally T4 and FSHvalues were assessed using the serum.

Additional groups were then designed to study the effects of thetreatment on a somatostatin receptor positive tumor. The first group wasdesigned to study the effects of treatment on large volume tumors(average volume 1720±608 mm³). This group, Cohort 5 (N=5), receivedthree fractionated doses of ²¹³Bi-DOTATOC (specific activity 7.4 MBq²¹³Bi/1 μG DOTATOC) with a total average cumulative dose of 13.0±0.5 MBqof ²¹³Bi-DOTATOC. The last cohort, the sixth cohort (N=4), was injectedwith two fractionated doses, ²¹³Bi-DOTATOC (specific activity 7.4 MBq²¹³Bi/1 μG DOTATOC) with a total average cumulative dose of 22.2±0.7 MBqof ²¹³Bi-DOTATOC. As described earlier, each dose was divided into 2injections separated by a 1 hour interval. Tumor response to thetreatment was assessed in all cohorts by daily tumor measurements.

A 26 week toxicology study was performed in 3 groups of rats. The firstgroup, Cohort 7 (N=3), received three fractionated doses ²¹³Bi-DOTATOC(specific activity 7.4 MBq ²¹³Bi/1 μG DOTATOC with a total averagecumulative dose of 12.75±1.1 MBq. The second group, Cohort 8 (N=3),received D-lysine (concentration 400 mg/mL) immediately before receivingthree fractionated doses ²¹³Bi-DOTATOC with a specific activity 7.4 MBq²¹³Bi/1 μG DOTATOC with a total average cumulative dose of 11.39±0.14MBq. The third group, cohort 9 (N=3) received 3 fractionated doses ofDOTATOC. Rats were injected with 2.56 μg, 0.5 μg, 0.5 μg DOTATOC, on day1, day 2 and day 3, respectively. As described earlier, each dose wasdivided into 2 injections separated by a 1 hour interval.

Pathology

Organs were harvested and immediately placed in 10% formalin for aminimum of 48 hours. Following fixation in formalin, bone samples wereplaced in decalcifying solution for 36 hours. Trimmed organs were sentto the TriCore Laboratories (Albuquerque, N.M.) where they were embeddedin paraffin, sectioned, and stained with Hematoxylin and Eosin (H andE). Histopathologic evaluation was performed by a board certifiedveterinary pathologist (DFK) who examined the following organs of eachanimal: heart, lung, kidneys, testicles, spleen, pancreas, pituitarybone marrow, urinary bladder, adrenals, and two different sections ofthe liver.

Sections of both the right and left kidneys were examined to determinenephrotoxicity in all cohorts. Bone marrow was examined to evaluatehypoplasia and other lesions in Cohorts 3 and 4. Interstitial nephritisand bone marrow were scored as follows: 0 no lesions, 1 minimal lesions,2 mild lesions, 3 moderate lesions, and 4 severe lesions.

Statistics

For the pathology scoring, to evaluate nephrotoxicity on the 6 treatmentgroups, a frequency analysis was performed in StatXact-5 using theJonckheere-Terpstra Test. For all other data, graphs and calculationswere performed in SiginaStat®-3 and SigmaPlot®-9 as well as Graph PadPrism®-4 using the t-test. For ail statistical tests results wereconsidered significant when p<0.05. Animal biodistribution and tumorvolume data are expressed as the a verage, plus or minus the SEM.

Results

The radiolabeling incorporation yields and radiochemical putity by ITLCand HPLC were greater than 99.9% and greater than 95% respectively.²¹³Bi-DOTATOC demonstrated acceptable stability and was unchanged after1 hour of in vitro incubation in rat serum. The biodistribution datademonstrated specific binding to sst (somatostatin) expressing tissues.Administration of free ²¹³Bi, compared to ²¹³Bi-DOTATOC, resulted inhigher accumulation of radioacti vity in non-tumor bearing rats at 3hours post injection in the kidneys (34.47±1.40% injected dose/gram vs.11.15±0.46%, p<0.0001), the bone marrow (0.31±0.01% injected, dose/gramvs. 0.06±0.02%, p<0.00023), the spleen (0.36±0.02% injected dose/gramvs. 0.08±0.01%, p<0.00053), the liver (0.50±0.05% injected dose/gram vs.0.14±0.02% p<0.002), the blood (0.07±0.01% injected dose/gram vs.0.02±0.00%, p<0.022), the testis (0.03±0.01% injected dose/gram vs.0.02±0.00%, p<0.016) and the stomach (0.25±0.00% injected dose/gram vs.0.08±0.01%, p<0.000015). Administration of ²¹³Bi-DOTATOC in tumorhearing rats versus non-tumor bearing rats showed a decreased uptake at1 hour in the pancreas (3.55±0.4% injected dose/gram vs. 1.44±0.05%,p<0.014) and the adrenals (3.55±0.57% injected dose/gram vs. 0.50±0.05%,p<0.0061) as shown in FIG. 13A-B.

No difference in creatinine clearance was seen between the control group(DOTATOC only) and the bismuth treated animals for the 25 day study.Hematology results also did not show any significant differences betweenthe control group and the bismuth treated animals. No significantchanges were found in the FSH values between treated and controlanimals. However, significance was seen with T4 values between the twohighest treatment groups 22.2 MBq (p<0.024) and 13.0 MBq (p<0.006) ascompared to control. (FIG. 14)

The results of the bone marrow analysts for the DOTATOC control groupversus the low dose (12.6 MBq) ²¹³Bi-DOTATOC treatment group showed nolesions at 25 days; neither hypoplasia nor hyperplasia was observed. Theaverage histopathologic score for nephrititis for each treatment groupwas <1. Representative kidney sections of the treated animals are shownin FIG. 15. Statistical analysis of the data showed that the likelihoodof interstitial nephrititis increased with increasing dose when alltreatment groups were analyzed (p<0.04 with the Jonckheere-TerpstraTest). This significance was lost when the high-dose treatment group(22.2 MBq) was eliminated Minimal toxicity was seen in the high-dosetreatment cohort, except for one kidney in this group, which showed mildinterstitial nephritis (Table 1).

Histopathological examination revealed ho evidence of treatment inducedtoxicity at 25 days in the heart, lungs, liver, spleen, and urinarybladder. No histopathologic abnormalities were seen in any of theanimals in the testes, adrenals, or pancreas. Pituitary cysts were seenin 2 out of 4 animals in the high-dose (22.2 MBq) treatment group.However, such cysts are generally considered to be incidental findingsin Lewis rats.

No difference in creatinine clearance was seen between the control group(DOTATOC only) and the bismuth treated animals for the 26 week study.Hematology results also did not show any significant differences betweenthe control group and the bismuth treated animals. No significantchanges were found in the FSH or T4 serum values between treated andcontrol animals.

Histopathological examination at 26 weeks found minimal nodular corticalhyperplasia in both adrenals in all ²¹³Bi-DOTATOC treated rats, whileonly 2 rats in the D-lysine cohort had one adrenal each with nodularcortical hyperplasia; no adrenal hyperplasia was seen in the controlcohort. Microcystic pancreatic degeneration, ranging from mild tomoderate was seen in all of the cohorts. Cardiomyopathy was seen in onerat in the ²¹³Bi-DOTATOC group, 2 rats in the D-lysine group, and norats in the control group. All groups contained some rats with mild tomoderate microcystic degeneration in the pituitary. In the Bi-DOTATOCcohort, 83% of the kidneys had minimal to mild interstitial nephritis,while the Lysine and the controls cohorts had 67%. All groups showedsome mild or moderate cholangiohepatitis and perivasculites in theliver. Animals in most groups, including the control groups had minimalto mild interstitial pneumonia.

A significant decrease in the rate of tumor growth was observed at 9days PI in small volume tumor bearing rats (0.75 mm²) treated withlow-dose ²¹³Bi-DOTATOC (12.6 MBq) as compared to controls (p<0.037)treated with only non-radioactive DOTATOC (FIG. 16). In the large-volumetumor bearing cohorts (1730 mm³), rats receiving high-dose (22.2 MBq)²¹³Bi-DOTATOC showed significant tumor reduction (approximately 3×) at 9days PI as compared to the rats receiving low-dose treatments (13 MBq)(p<0.025) (FIG. 17),

Tumor growth inhibition was observed in both small- and large-volumetumors when fractionated low-(12.6 MBq) and high-dose (22.2 MBq)²¹³Bi-DOTATOC were given, respectively. Previous reports indicate thatthe dose-limiting factor in PRRT is often nephrotoxicity caused by theradiation absorbed dose to the kidneys. Our results show only minimalnephrotoxicity observed with the low-dose ²¹³Bi-DOTATOC (Table 1) andmild nephrotoxicity was observed with high-dose ²¹³Bi-DOTATOC in onlyone animal. No significant changes in creatine clearance levels wereobserved in any of the treatment groups. The only evidence of othertreatment induced toxicities observed was a slightly lower T4 value inthe 13 MBq and the 22.2 MBq treatment groups at 25 days.

Enhancing Somatostatin Receptor Expression and Anticancer Therapy UsingFirst and Second Therapeutic Agents Example 1 Cytotoxicity ofRadiolabeled Somatostatin Analog

The somatostatin receptor (SSTr)-positive human pancreaticadenocarcinoma cell line Capan-2 was used as the test cell line, and theSSTr-negative human lung carcinoma cell line A549 was used as controlcell line. Radiolabeled somatostatin analogs ²¹³Bi-DOTATOC and¹⁷⁷Lu-DOTATOC were shown to be much more cytotoxic than non-somatostatinreceptor-specific DOTA and ¹⁷⁷Lu-DOTA in the somatostatin receptorexpressing cell line Capan-2 (FIG. 3). However, when ²¹³Bi-DOTATOC and¹⁷⁷Lu-DOTATOC cytotoxicity were compared with ²¹³Bi-DOTA and ¹⁷⁷Lu-DOTAcytotoxicity in somatostatin receptor negative cell line A549, nodifference was observed (FIG. 4). FIG. 3 shows the cytotoxic effects of37,000 becquerels (37 kBq) of ¹⁷⁷Lu and ²¹³Bi labeled to DOTA andDOTATOC on somatostatin receptor expressing cell line Capan-2. FIG. 4,on the other hand, shows the cytotoxic effects of 37 kBq of ¹⁷⁷Lu and²¹³Bi labeled to DOTA and DOTATOC on somatostatin receptor negative cellline A549.

Example 2 Effects of Gemcitabine Pre-Treatment on Somatostatin AnalogCytotoxicity

One μg/mL Gemcitabine HCl was added to a somatostatin receptorexpressing cell line (Capan-2) two hours prior to exposure toradiolabeled recognition ligand in order to evaluate the additiveeffects of gemcitabine when the cells were still exposed to gemcitabineand radionuclides. Note that this procedure is different fromradiosensitivity studies where gemcitabine is washed off before thereplenished cells are exposed to radiation. FIG. 5 shows the combinedcytotoxic effects of 37 kBq of radionuclide labeled to DOTA and DOTATOCin combination with 1 μg/mL gemcitabine pre-treatment on somatostatinreceptor expressing cell line Capan-2.

Example 3 Effects of Gemcitabine and Radiolabeled Somatostatin Analog onApoptosis

Alpha emitters such as ²¹³Bi are known to cause G₂M arrest and induceapoptosis in cancer cell lines. Experiments were thus performed toevaluate radiation-induced apoptosis and the radiobiological andradiotherapeutical relevance of this mode of cell death in selection ofa radionuclide for therapy. The results are provided in FIG. 6, whichshows the apoptotic effects of 37 kBq of ¹⁷⁷Lu and ²¹³Bi labeled to DOTAand DOTA TOC on the somatostatin receptor expressing cell line Capan-2.As expected, high-linear energy transfer (LET) alpha emitter ²¹³Biexhibited much greater induction of apoptosis compared to that of thelow-LET beta emitter ¹⁷⁷Lu. At 48 hours, ²¹³Bi-DOTATOC exhibitedapproximately 4 times greater induction of apoptosis than ¹⁷⁷Lu-DOTATOC, and 100 times greater induction of apoptosis than non-radioactiveDOTATOC.

FIG. 6 also shows the effects of incubation time. For example, with¹⁷⁷Lu-DOTATOC, significant induction of apoptosis was observed at 96hours, whereas almost similar induction of apoptosis was observed at 48hours for DOTATOC, suggesting faster and greater induction of apoptosisby ²¹³bismuth. Therefore, high-LET alpha emitter ²¹³Bi labeled toDOTATOC is generally preferred for induction of apoptosis as compared to¹⁷⁷Lu labeled to DOTATOC (p>0.09).

The effects of gemcitabine and a somatostatin analog on apoptosis areshown in FIG. 7. Activation or down regulation of pro- andanti-apoptotic genes influence cancer cell viability, cancer cellsensitivity to chemotherapy and radiotherapy, and tumor development andprogression. A variety of chemotherapeutic agents induce cell death viaapoptosis. Gemcitabine is known to induce apoptosis primarily due to Baxoverexpression, whereas Bcl-xl is known to reduce gemcitabine-inducedapoptosis. In contrast, Capan-2 is highly sensitive to Fas-mediatedapoptosis. FIG. 7 shows the additive apoptotic effects of 37 kBq ofradionuclide labeled to DOTA and DOTATOC in combination with 1 μg/mLgemcitabine pre-treatment on somatostatin receptor expressing cell lineCapan-2. The data is expressed as ±S.E.M. of mean apoptosis. As shown inFIG. 7, gemcitabine by itself does not induce any significant amount ofapoptosis. The same holds true for DOTATOC and gemcitabine combinations.But when combined with radiolabeled DOTATOC, a synergetk effect oninduction of apoptosis is observed. For ¹⁷⁷Lu-DOTATOC, this significantsynergetic effect is observed at 96 hours, whereas for ²¹³Bi-DOTATOCthis effect is observed at 48 more and is much more pronounced at 96hours.

Example 4 Gemcitibine Effect Somatostatin Receptor Expression andRe-Expression

A radioligand assay was performed to evaluate receptor binding by therecognition ligand after pre-treatmem of 4 days with 1 μg/mL gemcitabineand 4 days of replenishment. FIG. 8 shows increased ¹⁷⁷Lu-DOTATOCbinding to gemcitabine pre-treated Capan-2 cells. As shown in FIG. 8,treatment with gemcitabine resulted in significant overexpression ofbinding sites, with almost 70% more binding sites than the non-treatedCapan-2 cells.

The quantity of receptor ligand binding directly corresponds to theradiation dose gisen to the cells by the internalized ¹⁷⁷Lu-DOTATOC.When compared to non-treated Capan-2 cells, the radiolabeled DOTATOCdelivered around 150% more internalized dose to gemcitabine pre-treatedCapan-2 cells, as shown in Table 1, which shows the increased¹⁷⁷Lu-DOTATOC internalized dose of gemcitabine pre-treated Capan-2 cells

TABLE 1 Groups ¹⁷⁷Lu-DOTATOC % internalized Gemcitabine pre-treatedCapan-2 13.46 ± 0.033 cells Capan-2 cells  5.36 ± 0.053

The increased internalized dose to the cells would also influence thebiological response of the cells to the treatment. To evaluate thecorresponding biological response cell viability and apoptosis, assayswere performed, as described in Example 5.

Example 5 Biological Response to Gemcitabine Pre-Treatment

Effects of 37 kBq and 370 kBq of ¹⁷⁷Lu labeled DOTATOC and DOTA ongemcitabine pre-treated Capan-2 cells and non-treated cells wereevaluated using CellTiter-Glo® Luminescent Cell Viability Assay(Promega, Madison, Wisc.) and Cell Death Detection ELiSA^(PLUS) 10×(Roche Applied Sciences, IN) for apoptosis. Cell viability was evaluatedat 24 and 48 hours of incubations. FIG. 9 shows an increased¹⁷⁷Lu-DOTATOC effect on cell viability at 24 hours for gemcitabinepre-treated Capan-2 cells. From FIG. 9 at 24 hours, there were nosignificant effects observed for 70 pM and 7 fM DOTATOC on cellviability for gemcitabine pre-treated and non-treated Capan-2 cells.Effects of radiosensitivity induced by gemcitabine pre-treatment can beobserved at higher doses (370 kBq) of non-target specific ¹⁷⁷Lu-DOTA.For gemcitabine pre-treated Capan-2 cells, ¹⁷⁷Lu-DOTATOC has a muchpronounced and statistically significant effect on cell viability thannon-treated Capan-2 cells (p<0.06). This effect is better expressed athigher doses (370 kBq) of ¹⁷⁷Lu-DOTATOC, as demonstrated in FIG. 9.

FIG. 10 shows the increased effect of ¹⁷⁷Lu-DOTATOC on cell viability at48 hours for gemcitabine pre-treated Capan-2 cells. As illustrated inFIG. 10, similar kinds of effects were observed after 48 hours for 70 pMand 7 fM DOTATOC. After 48 hours of incubation, effects ofradiosensitivity induced by gemcitabine pre-ireatment were observed athigher doses (370 kBq) as well as lower doses (37 kBq) of non-targetspecific ¹⁷⁷Lu-DOTA, unlike the observations made after 24 hours ofincubation. At the same time, the gemcitabine pre-treatment effects ofhigher doses of ¹⁷⁷Lu-DOTATOC on cell viability was diminished comparedto non-treated Capan-2 cells, whereas the gemcitabine pre-treatmenteffects were more pronounced for lower doses of ¹⁷⁷Lu-DOTATOC, as shownin FIG. 10.

FIG. 11 shows an increased ¹⁷⁷Lu-DOTATOC effect on apoptosis at 72 hoursfor gemcitabine pre-treated Capan-2 cells. As shown by FIG. 11, DOTATOCwithout radiolabel had minimal effects on the induction of apoptosis.Enhancements of radiation induced apoptosis for non-target specific¹⁷⁷Lu-DOTA were observed for gemcitabine pre-treated Capan-2 cells as aresult of possible radiosensitive phenomenon. For gemcitabinepre-treated Capan-2 cells, ¹⁷⁷Lu-DOTATOC has a much pronounced andstatistically significant effect on apoptosis than non-treated Capan-2cells.

Summarizing the aspects of the invention provided by the examples,somatostatin receptor targeted radionuclide therapy using high-LETα-emitter ²¹³Bi and low-LET β-emitter ¹⁷⁷Lu labeled to DOTATOC showdecreased cell survival, increased cell killing, and induction ofapoptosis. ²¹³Bi-DOTATOC is significantly more potent in vitro due toits high-LET α-emission and enhanced effects on mitotic and apoptoticdeaths. Gemcitabine had overall additive and synergetic effects, withmodulation and overexpression of somatostatin receptor binding si tesafter 4 days of pre-treatment. Data provided in the figures used in theexamples above is generally expressed as ±S.E.M. of mean values.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and ammo acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference for all purposes. Anyinconsistency between the material incorporated by reference and thematerial set for in the specification as originally filed shall beresolved in favor of the specification as originally filed. Theforegoing detailed description and examples have been given for clarityof understanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A radiopharmaceutical composition comprising: a therapeutic agentcomprising ²¹³Bi; and a pharmaceutically acceptable carrier. 2-7.(canceled)
 8. The radiopharmaceutical composition of claim 1 wherein thetherapeutic agent comprises a peptide, a peptide analog, a peptidederivative or a peptidomimetic compound.
 9. The radiopharmaceuticalcomposition of claim 1 wherein the therapeutic agent is effective inpeptide receptor radionuclide therapy (PRRT). 10-12. (canceled)
 13. Aradiopharmaceutical composition comprising: an octreotide or analog orderivative thereof, comprising ²¹³Bi; and a pharmaceutically acceptablecarrier. 14-15. (canceled)
 16. The radiopharmaceutical composition ofclaim 13 wherein the therapeutic agent further comprises a targetingmoiety that targets the therapeutic agent to a selected mammalian tumorcell. 17-24. (canceled)
 25. A method of treating a subject afflictedwith a cancer or precancerous condition, comprising: administering afirst therapeutic agent to the subject, wherein the first therapeuticagent increases the expression level of somatostatin receptors in acancer cell; and administering a second therapeutic agent to thesubject, wherein the second therapeutic agent comprises: a recognitionligand that selectively binds to a somatostatin receptor on the cancercell; and a cytotoxic compound; so as to cause a deleterious effect oncancer cell.
 26. (canceled)
 27. The method of claim 25, wherein thecancer comprises a carcinoma, a sarcoma, or a lymphoma.
 28. The methodof claim 25, wherein the cancer is selected from the group consisting ofbreast, pancreatic, gastric, prostate, renal, colorectal, thyroid, lung,kidney, liver, central nervous system, and malignant lymphoid cancer.29. The method of claim 25, wherein the cancer comprises a cancer of theneuroendocrine or central nervous system.
 30. The method of claim 25,wherein the first therapeutic agent comprises a nucleoside analog.31-33. (canceled)
 34. The method of claim 30, wherein the firsttherapeutic agent or an active metabolite thereof comprises aribonucleotide reductase inhibitor.
 35. The method of claim 25, whereinthe cytotoxic compound comprises a radionuclide.
 36. The method of claim35, wherein the radionuclide is selected from the group consisting of⁹⁰yttrium, ¹¹¹indium, ¹⁷⁷lutetium, ²²⁵actinium, ²⁰⁹bismuth, ²¹²bismuth,²¹³bismuth, ⁶⁴copper, ⁶⁷copper, ⁷⁶arsenic, ⁷⁷arsenic, ²⁰³lead, ²⁰⁹lead,²¹²lead, ¹⁶⁶holmium, ¹⁵³promethium, ¹⁸⁶rhenium, ¹⁸⁸rhenium, and²¹¹astatine. 37-47. (canceled)
 48. A method of treating a somatostatinreceptor expressing cancerous tumor in a patient in need, the methodcomprising administering to said patient an effective amount of acomposition according to claim
 1. 49. The method according to claim 48wherein said cancerous tumor is a cancer of the breast, pancreas,stomach, prostate, kidney, colon, rectal, thyroid, lung, kidney, liver,brain/central nervous system and lymph (malignant lymphoid cancer).